Signaling pathways regulated by GSK3 in hematopoietic stem cells Abstract Hematopoietic stem cells (HSCs) possess two distinct features: the ability to self-renew and to differentiate into mature blood cells. The signaling pathways that regulate HSC self-renewal and differentiation are not well understood. Glycogen synthase kinase-3 (GSK3), a negative regulator of canonical Wnt signaling and a downstream target of hematopoietic growth factor signaling pathways that signal through Jak/Stat and PI3 kinase/Akt dependent pathways, has been implicated in the regulation of HSC activity. Canonical Wnt signaling has been implicated in HSC self-renewal, but the role of this pathway remains controversial. Recently, the PTEN-PI3K-mTOR pathway has been demonstrated to play an essential role in HSC maintenance. Our hypothesis is that GSK3 is a critical effector downstream of Wnt and PI3K within HSCs that regulates the HSC population. In support of this hypothesis, our preliminary data show that: 1) lithium and other selective GSK3 inhibitors significantly increase the number of HSCs in mice, 2) RNAi mediated depletion of Gsk3 in murine HSCs initially expands the HSC population in bone marrow transplants, but long-term inhibition of GSK3 exhausts HSC. 3) The expansion of HSCs by inhibition of GSK3 is mediated through 2-catenin while the depletion of HSCs is largely dependent on mTOR signaling. These observations support the hypothesis that GSK3 is an important regulator of hematopoiesis. In this proposal, we will extend our study to explore the functional role of GSK3 phosphorylation in HSCs. We will also address the isoform specific functions of Gsk3a vs. Gsk3b in hematopoietic cells with newly constructed conditional knockouts in mice. The non cell- autonomous effect of GSK3 inhibition on HSCs will also be explored with the conditional knockout of Gsk3 in the osteoblast niche. Furthermore, we will test whether modulating the activity of GSK3 and mTOR can expand HSCs in ex vivo culture. A functional screen to identify new regulators of HSCs self-renewal in ex vivo culture will be carried out. The overall goal of this project is to examine the respective contributions of Wnt and PTEN-PI3K-mTOR signaling in the response to GSK3 inhibition and to define the role of GSK3 in regulating these pathways within the hematopoietic system. The knowledge learned from these studies will provide new insights into the mechanisms of the regulation of HSC self-renewal and differentiation and may improve the therapy of hematopoietic disorders. 1